A new liquid chromatography/mass spectrometry method for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in urine

A new liquid chromatography/mass spectrometry method for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in urine
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   A new liquid chromatography/mass spectrometry method for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in urine Showket H. Bhat 1,2 , Stacy L. Gelhaus 1,2 , Clementina Mesaros 1,2 ,  Anil Vachani 2,3 , and Ian A.Blair  1,2,* 1 Center for Cancer Pharmacology, University of Pennsylvania School of Medicine, Philadelphia,PA 19104-4863, USA 2 Center of Excellence in Environmental Toxicology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-4863, USA 3 Division of Pulmonary, Allergy and Critical Care, University of Pennsylvania School of Medicine,Philadelphia, PA 19104-4863, USA  Abstract 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a carcinogenic nitrosamine producedupon curing tobacco. It is present in tobacco smoke and undergoes metabolism to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in the lungs. NNAL undergoes furtheruridine diphosphate glucuronosyltransferase (UGT)-mediated metabolism to give N- and O-glucuronide metabolites, which together with free (non-conjugated) NNAL are then excreted inthe urine. The ability to conduct validated analyses of free and conjugated NNAL in human urineis important in order to assess inter-individual differences in lung cancer risk from exposure tocigarette smoke. The use of stable isotope dilution (SID) methodology in combination with liquidchromatography/multiple reaction monitoring/mass spectrometry (LC/MRM-MS) provides thehighest bioanalytical specificity possible for such analyses. We describe a novel derivatizationprocedure, which results in the formation of a pre-ionized N  -propyl-NNAL derivative. Theincreased LC/MS sensitivity arising from this derivative then makes it possible to analyze freeNNAL in only 0.25 mL urine. This substantial reduction in urine volume when compared withother methods that have been developed will help preserve the limited amounts of stored urinesamples that are available from on-going longitudinal biomarker studies. The new high sensitivitySID LC/MRM-MS assay was employed to determine free and conjugated NNAL concentrationsin urine samples from 60 individual disease-free smokers. Effects of inter-individual differences inurinary creatinine clearance on NNAL concentrations were then assessed and three metabolizerphenotypes were identified in the 60 subjects from the ratio of urinary NNAL glucuronides/freeNNAL. Poor metabolizers (PMs, 14 subjects) with a ratio of NNAL glucuronides/free NNAL <2(mean = 1.3), intermediate metabolizers (IMs, 36 subjects) with a ratio between 2 and 5 (mean =3.4), and extensive metabolizers (EMs, 10 subjects) with a ratio >5 (mean = 11.1).Tobacco use is the single largest preventable cause of disease and premature death in theUnited States, accounting for at least 30% of cancer deaths. [1]  The nearly 40% reduction inmale lung cancer deaths between 1991 and 2003 has been attributed to smoking declines inthe last half century. [2]  4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), acarcinogenic nitrosamine produced upon curing tobacco, is present at relatively high Copyright © 2010 John Wiley & Sons, Ltd. * Correspondence to:   I. A. Blair, Center for Cancer Pharmacology, 854 BRB II/III, 421 Curie Blvd, University of Pennsylvania,Philadelphia, PA 19104-6160, USA. NIH Public Access Author Manuscript Rapid Commun Mass Spectrom  . Author manuscript; available in PMC 2012 May 09. Published in final edited form as: Rapid Commun Mass Spectrom  . 2011 January 15; 25(1): 115–121. doi:10.1002/rcm.4824. NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t    concentrations in tobacco smoke. [3]  Therefore, it has been suggested that NNK is animportant contributor to the etiology of lung and pancreatic cancer in smokers, oral cancer insmokeless tobacco users, and lung cancer in non-smokers exposed to environmental tobaccosmoke. [4]  The ketone that is present at C-1 of NNK is rapidly reduced to an alcohol in thelung primarily by aldo-keto reductases (AKRs) of the 1C family, [5]  carbonyl reductase 1(CBR1), and 11 β -hydroxysteroid dehydrogenase type 1 (HSD1) [6]  to give the carcinogenicNNAL metabolite (Fig. 1). NNK is rapidly converted into NNAL in the lung and thenactivated by CYP2A6 and CYP2A13 [7,8]  to hydroxylated reactive metabolites that alkylateDNA [9]  with the concomitant formation of keto alcohol and diol metabolites, respectively(Fig. 1). NNAL is also converted into its O  -glucuronide by UGT1A9, UGT2B7, andUGT2B17 [10,11]  and to its N  -glucuronide by UGT1A4 and UG2B10 (Fig. 1). [11,12]  The O  -and N  -glucuronide conjugates of NNAL are subsequently excreted in the urine. UrinaryNNAL has been widely used as a biomarker of exposure to the carcinogen NNK in tobaccosmoke, [4]  whereas the amount of conjugated NNAL has been suggested to provide an indexof how much detoxification has occurred. [13]  Therefore, the absolute amount of free NNALthat is excreted together with the ratio of glucuronide metabolites to free NNAL can beemployed as biomarkers to predict an individual’s risk of tobacco smoke-induced lungcancer. [14] Stable isotope dilution (SID) combined liquid chromatography/multiple reaction monitoring/ mass spectrometry (LC/MRM-MS) provides the most sensitive and specific methodologyavailable for the analysis of biomarkers. [15]  Under optimal operating conditions, theprecursor to product ion is monitored many times per second, resulting in extremelyreproducible chromatographic peak shapes. In this way, a heavy isotope labeled standard isused in SID LC/MRM-MS to establish the presence of a particular analyte using both the LCretention time and MS/MS mass selection of the triple quadrupole (TQ) instrument. Thislevel of specificity cannot be attained with any other bioanalytical technique. A number of SID LC/MRM-MS assays have been reported for the analysis of NNAL metabolites inurine [16–20]  and plasma. [21]  However, the analysis of free urinary NNAL is much morechallenging and requires larger volumes of urine – typically 5 mL. [16,20]  For longitudinalcancer etiology studies there are often limited volumes of relevant biofluids available and soit is necessary to improve the analytical sensitivity in order to reduce the volume of biofluidrequired. The concept of using pre-ionized derivatives to improve sensitivity has beenexploited for a number of different endogenous substances such as steroids [22–25]  andlipids. [26–28]  However, there is a paucity of reports on its utility for exogenously derivedanalytes. We found that the readily prepared pre-ionized (quaternary) N  -propyl derivative of NNAL increases the sensitivity of analysis by a factor of almost 20. We have used thisderivatization procedure coupled with SID LC/MRM-MS to validate an assay for NNALand its glucuronide metabolites in the urine of tobacco smokers. This has made it possible toexamine the relationship between the urinary concentrations of NNAL and its glucuronidemetabolites and creatinine excretion in limited urine volumes. The method was also used toestablish criteria for NNAL PM, IM, and EM phenotypes. EXPERIMENTAL Clinical study Single-void urine specimens were obtained from subjects enrolled in an ongoing study of tobacco biomarkers at the University of Pennsylvania School of Medicine. All samples werealiquoted and frozen at −80°C within 1 h of collection. They were then stored at −80°C untilanalysis. Major exclusion criteria for the study were history of malignancy or chronic lung,cardiac, renal, or liver disease, and current nicotine replacement use. A questionnaire wasadministered to all subjects that collected basic demographics, detailed tobacco history, anda focused medical history including current medication use. The study was approved by the Bhat et al.Page 2 Rapid Commun Mass Spectrom  . Author manuscript; available in PMC 2012 May 09. NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t    Institutional Review Board at the University of Pennsylvania, and all subjects gave writteninformed consent. Sixty urine samples were analyzed for free and conjugated NNAL. Materials 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and 4-(methyl-[ 2 H 3 ]-nitrosamino)-1-(3-pyridyl)-1-butanol ([ 2 H 3 ]-NNAL) were obtained from Toronto ResearchChemicals (Toronto, Canada). HPLC grade acetonitrile, ethyl acetate and ammonium acetatewere obtained from Fisher Scientific Co. (Fair Lawn, NJ, USA). Deionized water for thepreparation of mobile phases was produced in-house with a Milli-Q system (Millipore,Milford, MA, USA). Sodium phosphate monobasic, sodium phosphate dibasic, methyliodide, ethyl iodide, n  -propyl iodide and formic acid were purchased from Sigma-Aldrich(St. Louis, MO, USA). Disposable glass centrifuge tubes for extraction and derivatizationwere obtained from Kimble Chase (Vineland, NJ, USA). Liquid chromatography Chromatography was performed using a Waters HPLC system (Alliance 2690 Model,Waters Corporation, Milford, USA) equipped with a temperature-controlled autosampler.This was coupled with a TSQ Quantum Ultra triple quadruple mass spectrometer (TQ)(Thermo Electron) equipped with a heated electrospray ionization source (HESI). LC wasconducted using a Phenomenex Luna C 18  column (5 µm, 100 Å, 100 × 2 mm i.d.) precededby a guard column of the same packing material. Solvent A was aqueous 10 mM ammoniumacetate containing 1% acetonitrile and 0.01% (v/v) formic acid, and solvent B wasacetonitrile. The flow rate was 0.2 mL/min. A linear gradient was run as follows: 0 min, 0%B; 5 min, 0% B; 14 min, 100% B; 16 min, 100% B; 18 min, 0% B; and 28 min, 0% B. Thesamples (100 µL) were maintained at 4°C in the autosampler tray, and 20 µL of sample wasinjected for analysis. The gradient was started immediately after the sample injection. Mass spectrometry The mass spectrometer was tuned to its optimal sensitivity by directly infusing a solution of aqueous 1000 ng/mL of derivatized NNAL into the ion source via a syringe pump with acontinuous infusion of 50% acetonitrile containing 10 mM ammonium acetate and 0.01%formic acid (v/v) at a flow rate of 0.2 mL/min. The capillary temperature was 350°C, thespray voltage was 4500 V, and the vaporizer temperature was 350°C. Nitrogen was used forthe sheath gas and auxiliary gas set at 35 and 15 (arbitrary units), respectively. Data wasacquired in the MRM mode. Preparation of stock and standard solutions Solutions of NNAL and [ 2 H 3 ]-NNAL were prepared separately at a concentration of 1000ng/mL from respective parent stocks of 1 mg/mL in methanol through serial dilution inmethanol and kept frozen at −20°C. For spiking into the urine samples, [ 2 H 3 ]-NNAL wasprepared at a concentration of 5 ng/mL in water through serial dilution of 1000 ng/mLsolution. Similarly the highest calibration standard solution of NNAL at a concentration of 5ng/mL in water was prepared from the 1000 ng/mL solution by serial dilution. All othercalibration standard solutions were prepared from the highest calibration standard solutionby sequential dilutions with water. Quality control (QC) solutions were prepared again byserial dilution of highest calibration standard solution of NNAL (5 ng/mL) with water.NNAL QC solutions were prepared in NNAL-free urine at the lower limit of quantitation(LLOQ) of 20 pg/mL urine (0.10 nM); lower quartile quality control (LQC), 40 pg/mL urine(0.19 nM); mid-range quality control (MQC), 200 pg/mL urine (0.96 nM); and the upperquartile quality control (HQC), 800 pg/mL urine (3.83 nM). Pooled urine from a male non-smoker with no known ETS exposure was used to prepare all calibration standards and the Bhat et al.Page 3 Rapid Commun Mass Spectrom  . Author manuscript; available in PMC 2012 May 09. NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t    QCs. All standards and controls were frozen at −20°C until used. Free and conjugatedNNAL in this urine sample was below the limit of detection of the assay. Sample preparation for analysis by LC/MS Samples were analyzed for free and total NNAL separately in 0.25 mL urine. Urine samplesin 5 mL glass centrifuge tubes were spiked with 50 µL of 5 ng/mL [ 2 H 3 ]-NNAL (1 ng/mLfinal concentration) internal standard solution followed by addition of 200 µL of sodiumphosphate buffer (1 M, pH 6.8). For total NNAL analysis urine samples were treated with750 units of β -glucuronidase contained in 50 µL of water. Samples were capped and afterbrief vortex-mixing these were incubated at 37°C for 20 h with gentle shaking. Additionalunits of β -glucuronidase and/or an increase in incubation times did not lead to a significantincrease in the conversion of NNAL-glucuronides to free NNAL. Ethyl acetate (2 mL) wasadded, and the samples were extracted by vortex-mixing for 5 min. The two phases wereseparated by centrifugation at high speed for 5 min; the upper ethyl acetate layer wastransferred to 15 mL glass centrifuge tubes and dried under a N 2  stream. Dried urine extractswere then derivatized by addition of 100 µL acetonitrile/  n  -propyl iodide 80/20 (v/v). Tubeswere vortex-mixed, tightly capped and incubated at 60°C for 12 h. The reaction mixture wasthen dried under a N 2  stream and dissolved in 1% formic acid in water. Ethyl acetate (6 mL)was added, vortex-mixed, the tubes centrifuged, and the upper ethyl acetate layer wasdiscarded. The pH of the aqueous phase was increased to 8 by addition of 0.5 mL of 5%ammonium hydroxide in water and purified using a WCX (Waters) solid-phase extraction(SPE) column, which is a weak mixed-mode cation exchanger suitable for extraction of quaternary amines. Elution of the derivative was performed using 1 mL of 5% formic acid inmethanol. Purified extracts were dried in a speed vacuum concentrator and reconstituted in100 µL of starting mobile phase. Reconstituted samples were transferred to HPLC vials and20 µL injections were made for analysis. The MRM/MS transitions [M + ] 255.1 →  225.1(M + -NO) and 252.1 [M + ] →  222.1 (M + -NO) at a collision energy of 12 eV were used forthe N  -propyl derivatives of [ 2 H 3 ]-NNAL and NNAL, respectively. Method sensitivity Sensitivity of the method was evaluated by spiking four different concentrations – 50, 100,500 and 1000 pg/mL – in 0.25 mL urine. Evaluation was done by comparing the peak areasof NNAL after extraction and derivatization with NNAL which was extracted but notderivatized. Data analysis NNAL concentrations were determined from the ratio of the peak area of NNAL to the[ 2 H 3 ]-NNAL by interpolation from the standard curve. Quantification was performed usingX-caliber ®  software. Calibration curves were plotted using a linear regression. Eight NNALcalibration standards spanning from 20 to 1000 pg/mL were included in every sample setanalysis. In every sample set, QC samples were also run at four different levels that werechosen so as to match low, moderate and high levels of NNAL found in smoker urine. RESULTS AND DISCUSSION Liquid chromatography During the initial course of method development and validation, several different LCcolumns and relevant solvent systems were evaluated for the best chromatographicseparations of analyte from background interference. Poor peak shapes were observed onmost columns, except those with C18 and C8 stationary phases. However, if the startingmobile phase contained >2% organic modifier, poor peak shapes were also observed. Bhat et al.Page 4 Rapid Commun Mass Spectrom  . Author manuscript; available in PMC 2012 May 09. NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t    Therefore, the initial mobile phase was chosen as 100% 10 mM aqueous ammonium acetatecontaining 1% acetonitrile and 0.01% formic acid (v/v). The C18 column was held atambient temperature during gradient elution. Under these conditions, the C18 stationaryphase had not degraded significantly after >500 analyses. Mass spectrometry Using HESI in the positive ion mode, N  -propyl-NNAL and [ 2 H 3 ]- N  -propyl-NNALexhibited intense M +  at m/z   252 and 255, respectively (Table 1). CID and MS/MS analysisof M +  ( m/z   252) from N  -propyl-NNAL revealed product ions at m/z   222, 191, 163, 150,135, and 122 (Fig. 2(A)). The product ion spectrum of [ 2 H 3 ]-propyl-NNAL was quitesimilar to that obtained from the protium analog because only the M + –NO ion retained the[ 2 H 3 ]-methyl group (Fig. 2(B)). An almost identical series of product ions was observedpreviously for MH +  of NNAL. [20]  However, the proposed fragmentation pathway for MH + of NNAL would not account for the product ions observed in the pre-ionized N  -propylderivative. It seems likely that product ions derived from both protonated and N  -propylforms of NNAL actually arise through a remote charge fragmentation pathway. [29]  Theinitial fragmentation involves loss of an NO radical to generate a charge remote radicalcation (Figs. 2 and 3). This is followed by the loss of the methylamino group and cleavagealong the butyl backbone (Fig. 3). Confirmation of the proposed mechanism was obtainedby analysis of the methyl and ethyl derivatives of NNAL and [ 2 H 3 ]-NNAL (Table 1). Forquantitative LC/MRM-MS analysis of the N  -propyl derivatives of urinary NNAL, productions corresponding to loss of the NO radical at m/z   222 and 225 from the protium anddeuterium analogs, respectively, were employed. Internal standard The use of a deuterated internal standard is convenient as described previously by Pan et al  . [21]  However, it is not ideal because of the potential for deuterium exchange and theseparation of deuterium and protium analogs that are often observed during LCanalysis. [30–32]  Deuterium exchange could have been problematic because methanol wasused as stock solvent for NNAL and deuterated NNAL and acidic conditions were employedfor purifying the derivative from the urine. In spite of this potential problem no exchangewas observed when the internal standard was analyzed alone. We consistently found thatthat after NNAL derivatization, the protium contribution was <0.05%. Furthermore, therewas <0.02 min separation between the deuterium and protium forms of the NNALpropionate derivatives, which would minimize any differential suppression of ionizationbetween the analyte and its deuterated internal standard. [33–35]  Assay validation Standard curves were linear from 20 to 1000 pg/mL urine with typical r 2  >0.99 using linearregression. Over this range, validation of five replicate LLOQ, LOQ, MQC and HQCsamples on one day (Table 2) and three separate days (Table 3) met the criteria of precisionof 15% and accuracy between 85% and 115%. Typical chromatograms for a blank urinesample and the LLOQ samples are shown in Fig. 4. Similar chromatograms were obtainedfrom six other blank urine samples from non-smoking control subjects. A typical standardcurve is shown in Fig. 5. Stability analysis The stability tests were designed to match the expected conditions clinical samples mayexperience. The QC samples were stored at −80°C for 22 h and then taken out and kept atroom temperature for 2 h. This was repeated for three cycles of freezing and thawing. Nodegradation was observed for all four levels of QC samples and data were essentially Bhat et al.Page 5 Rapid Commun Mass Spectrom  . Author manuscript; available in PMC 2012 May 09. NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t  
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